Additive for livestok feeds

ABSTRACT

Methods and compositions are provided for method of enhancing feed utilization efficiency in a ruminant animal by adding to the feed a sufficient amount of a nonionic surfactant to enhance the utilization of the feed by the animal. The methods and compositions result in enhanced weight gain and/or milk production by the animal. Preferred nonionic surfactants include polyoxyethylenesorbitan monooleate and polyoxyethylenesorbitan trioleate at a concentration range of from about 0.01 to 1% (w/w) of the dry weight of the feed.

[0001] This application is a continuation-in-part of application Ser.No. 09/249,662, filed Feb. 12, 1999, which is a continuation ofapplication Ser. No. 08/872,654, filed Jun. 10, 1997, which is acontinuation of application Ser. No. 08/267,596, filed Jun. 28, 1994.

FIELD OF THE INVENTION

[0002] The present invention relates generally to ruminant feedcompositions containing nonionic surfactants, either alone or incombination with digestion enhancing agents, and to methods forenhancing feedstock utilization efficiency in ruminant livestock. Moreparticularly, this invention relates to the addition of nonionicsurfactants to ruminant feed, with or without added digestion enhancingagents, and feeding the resulting product to the animal.

BACKGROUND OF THE INVENTION

[0003] Anaerobic fermentation occurs during ruminant digestion, duringwhich proteins and carbohydrates are degraded. It is desirable inruminant digestion to be able to control protease and carbohydraseactivity to optimize the digestive process.

[0004] Since feed is a major cost in ruminant production, enhancingdigestive efficiency remains a driving objective in the industry.Although forages remain the major feed source, it is widely believedthat the efficiency of feed utilization by ruminants has remainedrelatively unchanged during the last two decades. New innovations thatenhance the digestive efficiency provide a compromise to emergingenvironmental concerns regarding ground water pollution in most dairyingareas. Nevertheless, an in depth understanding of the roles of feedprocessing and bacterial digestion are required to fully manipulate thedigestive processes of the rumen. Cheng et al. (“Microbial ecology andphysiology of feed degradation within the rumen,” in Physiologicalaspects of digestion and metabolism in ruminants: Proceedings of theseventh international symposium on ruminant physiology, Tsuda, Ed.,1991) has identified the following three general factors as influencingmicrobial digestion of feeds: (a) plant structures that regulatebacterial access to nutrients; (b) microbial factors that controladhesion and the development of digestive microbial consortia; and (c)complexes of oriented hydrolytic enzymes of the adherent microorganisms.Feed processing practices, e.g., grinding, normally attempt to increaseenzyme-substrate interaction by the exposition of susceptible substratebinding sites.

[0005] The manipulation of digestion within the rumen in order toincrease the efficiency of feed utilization has been achieved throughthe use of exogenous enzymes (Feng et al., “Effect of enzyme additiveson in situ and in vitro degradation of mature cool-season grass forage,”J. Anim. Sci. 70 (Suppl. 1):309 (1996)), and such compounds as ionophoreantibiotics, methane production inhibitors, inhibitors of proteolysis ordeamination, and buffers (Jouany, “Methods of manipulating the microbialmetabolism in the rumen,” Ann. Zootech. 43:49-62 (1994)). The increaseddigestive efficiency realized through the use of these compounds is theresult of major shifts in microbial fermentation pathways. For example,the selective use of antibiotics can alter the rumen microbialpopulation and ultimately influence the end products of digestion.Antibiotics are, however, used only in meat producing animals because ofthe risk of antibiotic transfer to milk. Production responses of animalsfed exogenous enzymes have been inconsistent. Exogenous enzymes havebeen shown to increase (Beauchemin et al., “Fibrolytic enzymes increasefiber digestibility and growth rate of steers fed dry forages,” Can. J.Anim. Sci. 75:641-644 (1995)), to not affect (Perry et al., “Effects ofsupplemental enzymes on nitrogen balance, digestibility of energy andnutrients and on growth and feed efficiency of cattle,” J. Anim. Sci.25:760-764 (1966)), and even to decrease (Svozil et al., “Application ofa cellulolytic preparation in nutrition of lambs” Sbor. Ved. Praci. VUVZPrhrelice 22:69-78 (1989)) the growth performance of ruminants fedforage or concentrate-based diets. The inconsistency is partly due tothe numerous enzyme preparations available, application methods, andtheir interaction with different types of diets.

[0006] Long-chain fatty acids and the halogen homologues of methane havebeen found to reduce methane production in the rumen (Van Nevel et al.,“Manipulation of rumen fermentation,” In: The Rumen MicrobialEcosystem., Ed. P. N. Hobson. Elsevier Applied Science, London, pp. 387et seq. (1988)). The reduction in methane production is usuallyassociated with a decrease in deamination of amino acids, particularly,branched-chain amino acids and an increase in propionic acid production.The main limitation with the use of these additives is that rumenmicrobes are able to adapt and degrade them after about one month oftreatment. Another disadvantage is that the beneficial effect appears tobe consistent only in forage-based diets that favor methane production.

[0007] Buffers are mainly used under conditions where the feeding ofhigh levels of grains can induce an active fermentation and cause excessproduction of acids within the rumen. They act by regulating andmaintaining the pH at levels at which the cellulolytic microorganismscan be of maximum effectiveness (pH=6-7). The digestion of starch andproteins is generally decreased when buffers are fed, however, theeffect on the digestion of cell wall carbohydrates is inconsistent(Jouany, “Methods of manipulating the microbial metabolism in therumen,” Ann. Zootech. 43:49-62 (1994)).

[0008] Surfactants have been used in the food processing industry asemulsifiers and extenders (Griffin et al., “Surface Active Agents,” inHandbook of Food Additives. 2^(nd) Ed., T. E. Furia, Ed., CRC Press, NewYork, N.Y., p 397 et seq. (1972)) and also as cleaning agents. The mostwell known physicochemical property of surfactants is their interfacialactivity when placed in solution. Their ability to align at theinterfaces is a reflection of their tendency to assume the mostenergetically stable orientation. One type of nonionic surfactant, thepolyoxyethylene sorbitan esters, are synthesized by the addition, viapolymerization, of ethylene oxide to sorbitan fatty acid esters. Thesenonionic hydrophilic emulsifiers are very effective antistaling agentsand are therefore used in a variety of bakery products. They are widelyknown as polysorbates. The effects of the polysorbate Tween 80 on thehydrolysis of newspaper was investigated by Castanon et al., “Effects ofthe surfactant Tween 80 on enzymatic hydrolysis of newspaper,”Biotechnol. & Bioeng. 23:1365 (1981). However, the effects of nonionicsurfactants on ruminant digestion have not heretofore been contemplated.

[0009] The present invention provides compositions and methods thatutilize nonionic surfactants to optimize the digestive process inruminant animals. The compositions and methods described in thisinvention enhance productivity of ruminant animals, reduce wasteproduction and ultimately improve profitability.

SUMMARY OF THE INVENTION

[0010] The present invention provides new and surprising methods andcompositions for enhancing feed utilization efficiency in ruminantanimals, such as cattle, sheep, goats, deer, bison, water buffalo andcamels. In particular, it has now been discovered that when nonionicsurfactants are admixed in ruminant feedstuffs at a concentration offrom about 0.01 to 1% (w/w) and the feedstuffs are fed to ruminants,significantly higher productivity can be expected from these animals.Higher productivity may be characterized by higher milk yield, increasedrate of weight gain, higher efficiency in converting feed into bodytissues or milk, and/or a reduction in manure production. No additionalbenefits are realized if excess amounts of the surfactant are admixedwith the feedstuff. It has also been discovered that when nonionicsurfactants at a concentration of from about 0.01 to 1% (w/w) arecombined with digestive enzymes, such as glycanases, and admixed withruminant feeds, ruminant animals consuming said feed have higher feedconversion efficiencies and productivity.

[0011] In other aspects, the present invention provides compositions andmethods that modify fermentation within the rumen towards more propionicacid production at the expense of acetic acid. Less heat is producedduring the metabolism of propionic acid in the animal compared to thatproduced during the metabolism of acetic acid. Therefore the methods andcompositions of the invention may be used to mitigate the effect of heatstress in ruminant animals.

[0012] In yet other aspects, the present invention provides methods forincorporating surfactant into ruminant feedstuff that ensures evendistribution of the surfactant in the feedstuffs in order to obtainconsistent improvement in animal performance. This aspect of theinvention extends to feed additives containing nonionic surfactantseither alone or in combination with digestion enhancing agents inconcentrations as specified in the present invention.

[0013] In one preferred embodiment of the present invention, a nonionicsurfactant is diluted with water or a carrier such as celite,diatomaceous earth, or silica and admixed with the feed before feedingthe feed to the animal. When diluted with water, the surfactant may besprayed onto the feed while the feed is simultaneously being mixed toensure even distribution of the surfactant in the entire feed material.The surfactant coats the surface of the feed to enhance attachment ofenzymes and or bacteria once the animal consumes the feed material.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The foregoing aspects and many of the attendant advantages ofthis invention will become more readily appreciated as the same becomebetter understood by reference to the following detailed description,when taken in conjunction with the accompanying drawings, wherein:

[0015]FIG. 1 is a graphical representation of the effect of the nonionicsurfactants polyoxyethylenesorbitan monooleate (Tween 60, shown as “”for protease activation (left axis) and “▪” for SH unmasking (rightaxis)), and polyoxyethylenesorbitan trioleate (Tween 80, shown as “♦”for protease activation (left axis) and “5” for SH unmasking (rightaxis)) as described in Example 2;

[0016]FIG. 2 is a graphical representation of the effect of the nonionicsurfactants Tween 60 (“▪”) and Tween 80 (5) compared to control (“♦”) onin vitro cellulose degradation as described in Example 2;

[0017]FIG. 3 is a graphical representation of the effect of the nonionicsurfactant Tween 80 on milk production in dairy cows as described inExample 4. In FIG. 3, ♦ represents the control, ▪ represents Tween 80 ata concentration of 0.2% w/w plus 0.1% enzyme, and 5 represents Tween 80at a concentration of 0.2% w/w;

[0018]FIG. 4 is a graphical representation of the effect of the nonionicsurfactant Tween 80 on milk production in mature dairy cows as describedin Example 4. In FIG. 4, ♦ represents the control, ▪ represents Tween 80at a concentration of 0.2% w/w plus 0.1% enzyme, and 5 represents Tween80 at a concentration of 0.2% w/w;

[0019]FIG. 5 is a graphical representation of the effect of the nonionicsurfactant Tween 80 on milk production in heifers as described inExample 4. In FIG. 5, ♦ represents the control, ▪ represents Tween 80 ata concentration of 0.2% w/w plus 0.1% enzyme, and 5 represents Tween 80at a concentration of 0.2% w/w;

[0020]FIG. 6 is a graphical representation of the effect of the nonionicsurfactant Tween 80 on milk production in fresh cows as described inExample 4. In FIG. 6, ♦ represents the control, ▪ represents Tween 80 ata concentration of 0.2% w/w plus 0.1% enzyme, and 5 represents Tween 80at a concentration of 0.2% w/w;

[0021]FIG. 7 is a graphical representation of the effect of the nonionicsurfactant Tween 80 at 0.2% (w/w) and 0.3% (w/w) concentration levels onmilk production in dairy cows as described in Example 5. In FIG. 7, ♦represents the control, ▪ represents Tween 80 at a concentration of 0.2%w/w plus 0.1% enzyme, and 5 represents Tween 80 at a concentration of0.2% w/w;

[0022]FIG. 8 is a graphical representation of the effect of the nonionicsurfactant Tween 80 at 0.2% (w/w) and 0.3% (w/w) concentration levels onmilk production in first calf heifers as described in Example 5. In FIG.8, ♦ represents the control, ▪ represents Tween 80 at a concentration of0.2% w/w, and 5 represents Tween 80 at a concentration of 0.3% w/w;

[0023]FIG. 9 is a graphical representation of the effect of the nonionicsurfactant Tween 80 at 0.2% (w/w) and 0.3% (w/w) concentrations levelson milk production in mature cows as described in Example 5. In FIG. 9,♦ represents the control, ▪ represents Tween 80 at a concentration of0.2% w/w, and 5 represents Tween 80 at a concentration of 0.3% w/w.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0024] According to one aspect of the present invention, methods andcompositions are provided for enhancing feed utilization efficiency inruminant animals, comprising adding to the feed a sufficient amount of anonionic surfactant to enhance the utilization of the feed by theanimal.

[0025] The term “feed efficiency” or “feed utilization” or “feedconversion” as used herein means the amount of feed needed to obtain agiven amount of weight gain or milk production. In particular, feedefficiency or utilization expresses the efficiency by which an animalconverts feed into weight gain or milk production. Feed efficiency isexpressed as the ratio of weight of feed to weight gain (or milkproduction).

[0026] Although the terms “feed efficiency” and “weight gain” are oftenused together, there is a significant difference between the two as canbe seen by the above definitions. Specifically, the determination offeed efficiency depends upon a given weight gain or milk productionwhereas the determination of weight gain or absolute milk productiondoes not depend upon a given feed efficiency. The differences areespecially significant to an animal producer or dairy farmer. Inparticular, weight gain or milk production can be achieved with little,no or even negative change in feed efficiency. Thus, for the animalproducer, merely obtaining increases in weight gain or milk productionmay not necessarily be a more cost effective method for growth of theanimal. While a producer looks at numerous factors in determining thecost of production, feed utilization efficiency is probably the mostimportant and has the most impact on cost per pound of meat produced.

[0027] Thus, in one aspect of the invention, methods and compositionsare provided for enhancing weight gain in a ruminant animal for a givenamount of animal feed, comprising adding to the feed a sufficient amountof a nonionic surfactant to enhance the weight gain by the animal. Inyet other aspects of the invention, methods and compositions areprovided for enhancing milk production by a ruminant animal, comprisingadding to the feed of the animal a sufficient amount of a nonionicsurfactant to enhance milk production by the animal. In still otheraspects of the invention, methods and compositions are provided forreducing the adverse effects of heat stress in a ruminant animal,comprising adding to the feed of the animal a sufficient amount of anonionic surfactant to enhance feed utilization efficiency, enhanceweight gain and/or enhance milk production by the animal.

[0028] As used herein, the term “ruminant” means an even-toed hoofedanimal which has a complex 3- or 4-chambered stomach, and which ischaracterized by chewing again what it has already swallowed. Someexamples of ruminants include cattle, sheep, goats, deer, bison, waterbuffalo and camels.

[0029] Surfactants include all surface active agents that are organic ororganic-metal molecules that exhibit polar and solubility behavior thatresult in the phenomenon known as surface activity. The most commonlyrecognized phenomenon in this respect is the reduction of the boundarybetween two immiscible fluids. Surfactants include surface active agentswhich act as emulsifiers, wetting agents, solubilizers, detergents,suspending agents, crystallization modifiers (both aqueous and nonaqueous) complexing agents and in other ways. The surfactants mostuseful in the practice of the present invention are the nonionicsurfactants, including, without limitation, polyoxyethylenesorbitanmonooleate (Tween 60), polyoxyethylenesorbitan trioleate (Tween 80),polyoxyethylenesorbitan monostearate, alkyltrimethylammonium bromides,dodecyltrimethylammonium bromide, hexadecyltrimethylammonium bromide,mixed alkyltrimethylammonium bromide, tetradecyltrimethylammoniumbromide, benzalkonium chloride, benzethonium chloride,benzyldimethyldodecylammonium bromide, benzyldimethylhexadecylammoniumbromide, benzyltrimethylammonium chloride, benzyltrimethylammoniummethoxide, cetylpyridinium bromide, cetylpyridinium chloride,cetyltributylphosphonium bromide, cetyltrimethylammonium bromide,decamethonium bromide, dimethyldioctadecylammonium bromide,methylbenzethonium chloride, methyl mixed trialkyl ammonium chloride,methyltrioctylammonium chloride, n,n′,mb′-polyethylene(10)-n-tallow-1,3-diamino-propane and 4-picoline dodecyl sulfate. In themost preferred form of the invention, the nonionic surfactant isselected from the group consisting of polyoxyethylenesorbitan monooleate(Tween 60) and polyoxyethylenesorbitan trioleate (Tween 80).

[0030] The concentration of surfactant affects the physical and chemicalproperties of the surface of feed particles, and consequently, digestionof the feed particle. During our earlier investigations, we determinedthat the range of concentrations of surfactants that promote associationof enzymes with feed particles is quite narrow. Insufficientconcentrations of surfactant did not increase interaction betweenenzymes and feed particles, whereas excess amounts tended to mask thesurface of the feed particles and impede enzyme attachment. For purposesof the present invention, ffective amounts of nonionic surfactants andtheir derivatives are from about 0.01 to 1% (w/w) of the dry weight ofthe feed, preferably from 0.01 to 0.5% (w/w) of the dry weight of thefeed, and most preferably from 0.01 to 0.3% (w/w) of the dry weight ofthe feed.

[0031] In one presently preferred embodiment of this invention, thenonionic surfactant is diluted with a suitable diluent that does notaffect the physico-chemical properties of surfactant before admixingwith feed for ease of application and to ensure that the surfactant isdistributed evenly in the feed. Suitable diluents include, but are notlimited to water, celite, diatomaceous earth, and silica.

[0032] Feedstuff or feed useful in the practice of the present inventionincludes forages and grain feeds, such as grass and legume forages, cropresidues, cereal grains, legume by-products and other agriculturalby-products. In situations where the resulting feed is to be processedor preserved, the feed may be treated with the surfactant and/or enzymebefore processing or preservation. Processing may include drying,ensiling, chopping, pelleting, cubing or baling in the case of forages,and in the case of grains and legume seeds by rolling, tempering,grinding, cracking, popping, extruding, pelleting, cubing, micronizing,roasting, flaking, cooking and or exploding.

[0033] As used herein, “forages” include the cut aerial portion of aplant material, both monocotyledonous and dicotyledonous, used as animalfeed. Examples include, without limitation, orchardgrass, timothy, tallfescue, ryegrass, alfalfa, sainfoin, clovers and vetches.

[0034] As used herein, “grain feeds,” means the seeds of plants that arefed to ruminant animals and may or may not include the outer hull, podor husk of the seed. Examples include, without limitation, corn, wheat,barley sorghum, triticale, rye, canola, and Soya beans.

[0035] The present invention may be combined with other feed processingtechniques or preservation methods, and may be included either duringprocessing or preservation. Other processing techniques useful incombination with the present invention include, but not limited to,drying, ensiling, chopping, grinding, pelleting, cubing or baling in thecase of forages, and in the case of grains feeds and legume seeds bydrying, rolling, tempering, grinding, cracking, popping, extruding,pelleting, cubing, micronizing, roasting, flaking, cooking and orexploding. Preservation may include, but not limited to ensiling andhaymaking.

[0036] Surfactants and enzymes used in accordance with the presentinvention are available in either a liquid or powdered form. If a liquidis used, the surfactant may be sprayed “as is” onto the feed material orpreferably diluted in the same or separate aqueous solutions beforeapplication. When provided as a nonionic surfactant coated on a solid,the surfactant preferably may constitute at least 50% of the dry weightof the product. If provided as a solid it may be applied to the feedmaterial “as is” or preferably dissolved in water or aqueous solutionssuch as a buffer solution with a pH range from 4.5 to 7 beforeapplication.

[0037] The surfactant is then evenly applied to the feed material. Theresulting feed can either be fed immediately to livestock or stored andfed at a later time. The resulting feed composition is effective forprolonged periods of time, such as for at least three years or longerdepending on the nature of the feed composition, storage conditions andthe like.

[0038] In addition to feed and a nonionic surfactant, the compositionsof the invention may further comprise one or more additional agents thatenhance the ruminant digestive processes. Such agents include, forexample, pyrodoxal 5-phosphate, fumaric acid and its salts, sorbic acidand its salts, parabenzoic acid esters, benzoic acid, polydimethylsiloxane-polyethers, unsaturated alcohols, bentonite, proteolytic and/orcarbohydrase enzymes, such as glycanase, hemicellase, cellulase,pectinase, xylanase and amylase, and lactic acid bacteria inoculants,such as those comprising Lactobacillus casei, L. acidophilus, L.salivarius, L. corymiformis subsp coryniformis, L. curvatus, L.plantarum, L. brevis, L. buchneri, L. fermentum, L. viridescens,Pdiococcus acidilacti, P. cerevisiae, P. pentosaceus, Streptococcusfaecalis, S. faecium, S. lactis, L. buchneri, L. fermentum, L.viridenscens, L. delbrueckiin, Leuconostoc cremoris, L. dextranicum, L.mesenteroides or L. citrovorum. Where the surfactant is used inconjunction with exogenous glycanases, the method of producing feedcompositions in the present invention is most effective when surfactantconstitute on the order of about 0.01% of the dry weight of the feed. Insituations where the surfactant is used without exogenous enzymes, thecompositions are most effective when the surfactant concentration doesnot exceed about 0.2% of the dry weight of the feed.

EXAMPLE 1 Protease Activity and Adsorption

[0039] Animals, feed and rumen fluid collection

[0040] Two rumen-fistulated, nonlactating cows averaging 623±12.5 kg inweight were fed 5 kg dry matter (DM) of low quality timothy hay twicedaily. About 2.5 L of rumen fluid was collected through the fistula 4hrs after the morning feeding at 07:00 hrs. Bulk feed particles wereremoved by sieving the fluid through a 0.5 mm strain. The fluid was thencomposited and then stored in a prewarmed (37° C.) thermal container.

[0041] Preparation of rumen mixed microbial cell and enzyme source

[0042] A microbial powder was prepared using the acetone-butanolextraction procedure outlined by Mahadevan et al., “Preparation ofprotease from mixed rumen microorganisms and its use for the in vitrodetermination of true protein in feedstuffs,” Can. J. Anim. Sci. 67:55(1987). About 500 g of this powder was prepared and stored at −20° C.Extraction of the proteases was accomplished by stirring 250 g of thepowder with 1 L of 4° C. cold water (for 1 hr) and then proceeding alongMahadevan's extraction procedure. Only extracts from the filtration withan XM-300 Amicon Filter, (approx. 300 000 molecular weight cutoff—undernitrogen gas), were made, washed twice with distilled water and theretentate freeze dried. This was referred to as the mixed microbial cellenzyme source. It was used in the protein adhesion tests and also in theparallel thiol and protease activity determinations.

[0043] Determination of thiols and protease activity and bacterialprotein adhesion

[0044] Ten grams of the mixed microbial cell enzyme was dissolved in 100ml warm (37° C.) 0.1 M phosphate buffer, pH 6.8, and used as an enzymeinoculant. The assay matrix consisted of 1 ml enzyme source, 1 ml 2%casein solution, 1 ml 01 M phosphate buffer and 1 ml of either therelevant level of surfactant or an equivalent amount of buffer. Tenlevels of the two surfactants, polyoxyethylene sorbitan monoleate (Tween80) and polyoxyethylene sorbitan monastearate (Tween 60) were tested,viz 0, 0.08, 0.16, 0.24, 0.32, 0.4, 0.8, 1.2, 1.6, and, 2.0% surfactantin the assay mixture.

[0045] The protease activity incubations were performed in 50 ml plasticcentrifuge tubes, at 37° C. and under a stream of carbon dioxide gas.After 1.5 hr, 1 ml of the assay mixture was removed for thedetermination of thiols (SH) and disulfides (SS) (Sasago et al.,“Determination of sulfhydryl and disulfide groups in milk byp-chloromercuribenzoate-diathizone method,” J. Dairy Sci. 46:1348-1351(1963)). At the end of 2 hr incubation, the reaction was stopped with 1ml of 15% TCA (trichloroacetic acid) solution, cooled to 4° C. under anicebath, and centrifuged at 10,000 for 10 min. The free amino acids inthe supernatant were assayed using the ninhydrin method (Rosen, “aModified Colorimetric Analysis For Amino Acids,” Arch. Biochim. Biophys.67:10 (1957)). The optimal surfactant inclusion level was calculated bydirect linear plots on the assumption that the Michaellis-Mentenequation applied.

[0046] For the cellulose adhesion tests, the microbial enzyme source wasresuspended and tested for adhesion on to a cellulose substrate (barleystraw, with 4% CP and ground through 0.5 mm sieve). Microbial adsorptionwas demonstrated by stirring, (120 strokes/min) at 30° C. 0.1 g of thestraw in 5 ml of a bacterial cell-enzyme inoculum suspension, and thenfollowing the supernatant protein change with time. Readings were takenat 10, 20, 30, 60, and 120 min. At the end of the adsorption period, theassay contents were centrifuged at 2500 g max for 10 min to precipitatethe solids, and the protein in the supernatant was precipitated out by15% TCA solution and quantified by the Bicichoninic method (Smith etal., “Measurement of protein using bicinchonic acid,” Anal. Biochem.150:76 (1984)). The mother suspension contained 4.0 g of the lyophilizedmixed bacteria and enzyme in 400 ml of 0.1 M phosphate buffer pH 6.8,with the following levels of surfactant (Tween-80); 0, 0.1, 0.25, and0.5%.

EXAMPLE 2 In Vitro Protein and Cellulose Degradation

[0047] Preparation of rumen fluid inoculum

[0048] A bacterial fraction, largely free of protozoa, was prepared forthe fermentation assays by using the procedure of Forsberg, “SomeEffects of Arsenic on the Rumen Microflora; An In Vitro Study,” Can. J.Microbiol. 24:36 (1978). The digesta inoculum was resuspended and washedtwice in an equivalent amount of 0.1 M phosphate buffer pH 6.8 to therumen fluid. The inoculum provided both the substrate and the enzymeused for cellulose degradation assay. Incubation periods were 0, 1.5, 3,6, 12, 24, and 48 hr. Other incubation conditions were similar to thoseoutlined in experiment 1 above. However, cellulose was determined by themethod of Updergraff, supra, for the in vivo digestibility trial. Theoptimal level of Tween 80 obtained was adopted here and followed.

[0049] In vivo digestibility trial

[0050] Four wethers weighing (72.5±15.0 kg) fitted with both rumen andduodenal cannula, were offered chopped medium quality timothy hay adlibitum. The hay was either sprayed with 500 ml water or 50 ml of Tween80 dissolved in 500 ml of water. The feed was offered in two equalportions, at 08:30 and 20:30 hr. Water was available ad libitum. Theexperiment was designed as a 2×2 latin square with two 14-dayadaptation, two 7-day collection periods.

[0051] Estimates of the rates of passage of the two treated hays weremade using chromium mordanted fibre (Cr) for the particulate phase andcobalt-ethylene diaminetetracetic acid (Co-EDTA) for the liquid phase.The method of Uden et al., “Investigation of chromium, cerium and cobaltas markers in digesta: Rate of Passage studies,” J. Sci. of Food andAgriculture 31:625-632 (1980), was used in the preparation of bothmarkers.

[0052] The sheep were adapted to the feed in individual pens, and thenmoved into digestibility cages for total collection and marker infusion.During the collection period, records of feed intake, faecal and urineoutput were maintained. 250 g subsamples of the faeces were collecteddaily, subsampled for DM determination, while the rest was dried under adrought oven. Urine was collected under 1N sulphuric acid.

[0053] On the last day of the collection period, each sheep was given 50g of Cr-mordanted fibre 1 hr prior to the evening feeding. In addition,250 ml of Co-EDTA (0.1 g/ml) was infused intraruminally and the animalswere then fed. Rumen digesta and duodenal sampling commenced 4 hr afterdosing and continued at the same interval for 96 hr.

[0054] Two samples of rumen fluid were collected: The first rumen fluidsamples (30 ml) were preserved for microbial protein estimations byadding 7.5 ml of 0.9% NaCl in 37% formaldehyde solution. The sampleswere then stored at −20° C. after the preparation of a bacterial pelletby centrifugation at 27,000 g max for 15 min. A portion of the secondrumen fluid and faecal samples was dried at 80° C. and ground in acoffee grinder (Braun, Inc. Mass.) for DM and Cr determination (Uden etal., 1980). Cr concentration in the samples was determined in duplicateby atomic absorption spectrophotometer (Perkin Elmer 560) using Crstandards (Fisher Scientific Co. N.J.). The rest of the second rumenfluid sample was centrifuged at 10,000 g max for 10 min and thesupernatants analyzed directly for cobalt using 0.1N HCL as the blank.Standards were prepared using cobalt chloride (Fisher Scientific Co.N.J.). Marker concentrations were expressed per gram of dry sample.

[0055] Other analyses included: acid and neutral detergent fibre(Goering and Van Soest, “Forage fiber analysis,” Agric. Handbook No.379, p. 12 (1970)), total N (Parkinson and Allen, “A wet oxidationprocedure suitable for the determination of nitrogen and mineralnutrients in biological materials,” Comm. Soil Sci. Plant Anal. 6:1(1975)) for RNA concentrations of the rumen bacterial pellets andduodenal samples.

[0056] Results

[0057]FIG. 1 shows the effect of Tween 60 and 80 on the activation ofrumen microbial proteinases and the unmasking of the reactive cysteine(SH) groups. The initial rates of proteinase activation were, 163.5%(s.e. 14.69) and 98.04% (s.e. 0.13) control=0. The optimal surfactantinclusion level was calculated by direct linear plots on the assumptionthat the Michaelis-Menten equation applies. The concentration ofadditive required to achieve half the maximum velocity of the rumenenzyme, provided that the protein substrates were at saturatingconcentrations is given by the Km value. Vmax values represent thevelocity of the enzyme reaction when all substrates are at saturatingconcentrations. The protease activation rate (Vmax) due to Tween 80 wassignificantly higher than that of Tween 60 (Table 1). Further theconcentration (Km) of Tween 80 required to elucidate this effect wasalso significantly lower than in Tween 60. TABLE 1 The apparentcoefficients of proteinase activation and SH unmasking Km¹ Vmax²Proteinase activation Tween 60 0.28 ± 0.02a  99.2 ± 2.7a Tween 80 0.20 ±0.03b 166.8 ± 8.9b Max. rate (μmol SH/mg protein/ SH unmasking % changein surfactant) Tween 60 0.30 ± 0.03a Tween 80 0.98 ± 0.29b

[0058] Column values followed by similar letters are not significantlydifferent (P<0.05). ¹Additive conc. (%). ²Maximum proteinase activation(%/unit additive conc.).

[0059] The effect of either Tween 60 or 80 on rumen cellulase activityis depicted in FIG. 2. 0.25% of either surfactant was used in thereaction mixture, based on the results in Table 1, (approximately Kmvalue). The rates of cellulose breakdown calculated by regressionanalysis on the initial 24 hr incubation period are shown in Table 2.The results show that the addition of either surfactant increased therate of cellulose breakdown significantly (P<0.05). TABLE 2 Initialrates of cellulose degradation. Treatment rate (μg/ml/hr) no additive0.60a (0.21) Tween 60 0.87b (0.28) Tween 80 1.04c (0.32)

[0060] Column values followed by similar letters are not significantlydifferent (P<0.05). Bracketed values are standard errors.

[0061]FIG. 3 shows the effect of Tween 80 on rumen microbial cellsenzyme source adsorption to barley straw over time. The addition ofTween 80 significantly (P<0.05) increased microbial protein adsorptionlevels greater than 0.1% did not alter either the rate or the extent ofadsorption significantly (P<0.05), (Table 3). The effect of Tween 60 onmicrobial protein adsorption to ground straw was not determined. TABLE 3Coefficients of microbial protein adsorption to cereal straw Microbialprotein adsorption Additive level (%) rate (μg/mg/min) extent (μg/mg) noadditive 0.026a (0.03) 0.94a 0.05% Tween 80 0.032b (0.01) 1.12b 0.10%Tween 80 0.034c (0.02) 1.21c 0.25% Tween 80 0.035c (0.01) 1.18c 0.50%Tween 80 0.034c (0.02) 1.19c

[0062] Column values followed by similar letters are not significantlydifferent (P<0.05). Bracketed values are standard errors.

[0063] On the basis that the in vitro results showed a potentialpositive response to rumen digestibility, an in vivo trial was carriedout. Table 4 shows the chemical composition of the hay used. TABLE 4 HayComposition (% by weight) % DM % CP % NDF % ADF % ASH Hay 0.82 11.8 68.333.85 3.4

[0064] Table 5 shows the digestibility coefficients of the two rationsused in the in vivo trial. The concentration of Tween 80 in the hayration was tested against a control. TABLE 5 Intake and digestibilitycoefficients from the sheep trial. Control Tween 80 Intake¹  1.96a (0.2) 2.06b (0.1) Digestibility; Dry matter (%) 54.46a (0.7) 64.70b (0.8)Crude protein (%) 52.44a (0.6) 61.25b (0.5) Acid detergent fibre (%)45.09a (0.5) 52.68b (0.4) Neutral detergent fibre (%) 50.13a (0.7)60.63b (0.7)

[0065]¹expressed as a % of the body weight. Values in a row followed bydifferent letters are significantly different (P<0.05). Bracketed valuesare standard errors.

[0066] From FIG. 1, is evident that either of the two surfactantsincreased rumen bacterial proteinase activity significantly. Incomparison with Tween 60, Tween 80 would have a higher solubilizingcapacity as a result of its slightly higher hydrophile-lipophile balance(HLB). The HLB for Tween 60 and Tween 80 are 14.9 and 15, respectively(Griffin et al., 1972).

[0067] It is tempting to attribute the gains in proteolysis wholly toincreased enzyme access resulting from both SH unmasking and increasedsubstrate solubility. However, higher levels of surfactants (<0.5%)would be required to achieve this. In spite of surfactant concentration,significant increases in proteolytic activity were observed at lowlevels (0.05-0.4%) of surfactant. The rate of SH unmasking was notsignificant at these points. Hence, in addition to solubility mediatedSH unmasking, a different mechanism of activation must be involved,particularly at low surfactant levels. Although the mechanism of actionseems unclear, it is possible that surfactant lipids would provide sitesfor enzyme-substrate hydrophobic interaction. Since the SH groups ofmost cysteine proteinases are located in hydrophobic environments withinthe enzymes molecules, nonionic surfactants would further enhanceinteraction with potential substrates.

[0068] The apparent Michaelis-Menten coefficients in Table 1 shows thatfor purposes of enhancing rumen proteinase function, Tween 80 would bepreferred to Tween 60. Further to obtain the same activation rate(Vmax), much less Tween 80 would be needed compared to Tween 60 as isshown by a lower Km value for the former additive. Obviously, in vivobenefits would only be made if increased proteinase activity is coupledto significant increases in fibre fermentation and ultimately toenhanced nutrient digestibility.

[0069] Table 2 shows that both additives enhanced cellulose degradationrate compared to the control treatment. However, the effect due to Tween80 was significantly greater than with Tween 60. Nonionic surfactantsare widely used in industrial bioreactors, to enhance cellulosehydrolysis.

[0070] The effects of various levels of Tween 80 on the microbial enzymesource adsorption to finely ground straw are summarized in Table 3.Although 0.05-0.10% Tween 80 in the reaction mixture increased microbialenzyme source adsorption significantly (P<0.05), the effect was notadditive at 0.25 or 0.50%. The adsorbing protein comprised ofproteinases, cellulases, other enzymes and unlysed bacterial cells.However, the adsorption of cellulases usually parallels the rate ofhydrolysis of cellulose. Hence, increased cellulase attachment at 0.5%Tween 80 may have contributed to the significantly higher rate ofcellulase degradation shown in Table 7.

[0071] Table 4 shows the chemical composition of the hay fed to sheep inthe trial designed to evaluate the effect of Tween 80 on intake anddigestibility. A medium quality hay was used so that protein would notlimit rumen function. As the results of this trial show (Table 5), Tween80 enhanced feed intake and digestibility significantly compared to thecontrol. There was a 5% and an 18% increase in intake and digestibility,respectively. It should be noted though that Tween 80 was included atabout 0.3% in the ration. However, this was the Km concentration that ishalf the concentration that would elicit maximum microbial activity.Consequently, the resultant effect on digestibility would be lower thanthe potential.

[0072] The observed increased digestion efficiency noted above was alsocoupled to increased feed intake. Normally, increased feed intake isalso associated with a more rapid digesta flow rate and a subsequentreduction in digestibility. That both intake and digestibility increasedtogether, reflects the increased efficiency of the digestive enzymes,particularly in the rumen.

EXAMPLE 3 Carriers For Tween 80

[0073] The specific objective of this experiment was to select a carrierthat will permit Tween 80 to be handled as a solid material rather thana liquid. In its natural form Tween 80 has a consistency similar tomolasses and this causes concern over mixing, particularly in coldweather. Three carriers (approved for use in the feed industry) wereidentified and evaluated as outlined below.

[0074] The carriers were celite (Fisher Scientific Co. New Jersey, USA),diatomaceous earth (Sigma Chemical Co. St. Louis, Mo.) and LuctaCarrier(Lucta, S.A. Barcelona, Spain). Tween 80 was mixed with the carrierssuch that the resulting mixture contained 50% Tween 80 (wt/wt). Theability of Tween 80 in these mixtures to improve digestive efficiencywas evaluated in vitro with orchardgrass hay that had been ground topass through a 1 mm screen. Treatments included 0 (control), 0.1 and0.2% liquid Tween 80, 0.1 and 0.2% Tween 80 in diatomaceous earth, 0.1and 0.2% Tween 80 in celite, and 0.1 and 0.2% Tween 80 in LuctaCarrier.Appropriate quantities of each substrate were mixed with the additivesand incubated in the Ankom in vitro system (Ankom Technology FairportN.Y.) for 22 h.

[0075] In vitro true digestibility (IVTD) of orchardgrass hay was higher(P<0.05) in all treatments containing Tween 80 except the treatmentcontaining 0.2% Tween 80 in LuctaCarrier. The IVTD values for control,and 0.1 and 0.2% Tween 80 in liquid form, 0.1 and 0.2% Tween 80 inLuctaCarrier, 0.1 and 0.2% Tween 80 in diatomaceous earth and 0.1 and0.2% Tween 80 in celite were: 51.44; 54.20, 54.93; 53.64, 49.45; 54.91,55.34; 54.41, 55.63%, respectively. These results indicate that all thecarriers investigated were equally effective as a means of deliveringthe Tween 80. The results further indicate that 0.1% (wt/wt) Tween 80may be as effective as 0.2% Tween 80 in increasing the extent of invitro true digestibility of orchardgrass hay.

EXAMPLE 4 Addition of Tween 80 to a Total Mixed Ration (TMR) Based onSilage and Barley Grain Improves Milk Production in Dairy Cows

[0076] One hundred and twenty cows and heifers in a dairy herd ofHolsteins were divided into three treatment groups of 40 animals pergroup. All animals were given ad libitum access to a total mixed ration(TMR) based on grass silage, corn silage, grass hay, barley and canolameal. The treatments imposed were:

[0077] Treatment 1—TMR without additive (Control).

[0078] Treatment 2—TMR formulated to contain 0.2% (wt/wt) Tween 80+0.1%enzyme preparation (wt/wt).

[0079] Treatment 3—TMR formulated to contain 0.2% (wt/wt) Tween 80.

[0080] The Tween 80 was coated onto silica to form a product containing50% Tween 80 and 50% silica. The enzyme preparation was obtained fromLucta S.A. (Barcelona, Spain). The preparation had the followingactivities: β-glucanase 263.0, xylanase 75.1 and amylase 542.6.Activities were expressed as nmol of reducing sugars released per mg ofenzyme in 1 min at 0.83 mg/ml enzyme concentration. The trial lasted 13weeks. Animals received their respective dietary treatments for 12weeks. Milk production and feed intake were monitored until the 13thweek (1 week after animals had been removed from dietary treatments).

[0081] Feed (TMR) offered to each group was weighed and recorded at eachfeeding. Each group was fed to provide a weighback of 5%. Samples of thefeed offered and refused were taken daily, composited into weeklysamples and dried at 55° C. for 72 hr to determine dry matter (DM)content. Daily (AM and PM) milk production by each cow was recorded.Animals were weighed two days in a row immediately after milking on amonthly basis. Milk samples were taken for compositional analyses (fat,protein, and somatic cells) in the week preceding the trial, and thenduring the trial at 4 week intervals. Samples were taken from both theAM and the PM milkings and analyzed individually.

[0082] The overall milk yield for cows that were lactating at least 3weeks prior to the start of the trial is presented in FIG. 4. Milkproduction from cows that received the combination of Tween 80 plusenzyme treatment was higher than the controls at all times. The upperrange of the difference was close to 2 kg/cow/day. The average increasewas 0.96 kg/cow/day. Over the 12-week period when cows were on theirrespective dietary treatments, a cow on the Tween 80 plus enzymetreatment produced 81 kg more milk than a cow on the control treatment.Compared to the control treatment, milk production was also higher incows that received the Tween 80 alone treatment. The average improvementin milk production from Tween 80 only over the trial was 0.76kg/cow/day. On average, a cow on Tween 80 produced a total of 64 kg moremilk during the 12-week period than a cow on the control diet. Theaverage increase in production of mature cows on Tween 80 alone was 1.31kg/day (FIG. 4). Over the 12 weeks of the trial, a mature cow receivingTween 80 alone produced 110 kg more milk, than a cow on the controldiet, and 74 kg more than a cow receiving the treatment containing Tween80 plus enzyme. There was a much larger response to the Tween 80 plusenzyme combination in heifers (FIG. 5). This response increased as thetrial progressed. Average increase in milk production in heifersreceiving the combination of Tween 80 plus enzyme was 2.6 kg/day abovethat of controls.

[0083]FIG. 6 shows the response of fresh cows (7 animals per treatmentgroup) to the dietary treatments. As indicated in the figure, there wasapparently no response to the dietary treatments prior to the 4th weekof lactation. After the 4th week, cows on the Tween 80 only treatmentproduced approximately 4 kg more milk/cow/day than cows on the controltreatment. The respective response by cows on the combination of Tween80 and enzyme treatment was 2 kg more milk/cow/day. The dietarytreatments did not affect milk composition (fat and protein) and somaticcell counts.

[0084] Weight Gain

[0085] Feed conversion efficiency was higher in animals that receivedTween 80 in their ration. Milk produced (kg) per kg of feed consumed was1.37, 1.40 and 1.48 for animals on the control, Tween 80 plus enzyme,and Tween 80 only treatments, respectively.

[0086] The average daily gain in weight of animals on Tween 80 plusenzyme treatment was higher than that of animals on the controltreatment. Weight gain of animals on the Tween 80 alone was similar tothat of animals on the control diet. This indicates that the additionalmilk produced by animals on Tween 80 plus enzyme, and Tween 80 onlytreatments was not derived from body tissue. In terms of energeticefficiency these animals were obtaining more from the diet than those onthe control treatment were.

EXAMPLE 5 Effect of two levels of Tween 80 on milk production and feedintake in cows

[0087] Seventy-five dairy cows of the Holstein breed were rankedaccording to lactation number, days in milk and production level andplaced into three equal groups. Treatments were then randomly assignedto individual animals within the groups. There were 25 animals in eachdietary treatment group. Cows were offered ad libitum access to a totalmixed ration (TMR) based on grass silage, corn silage, grass hay and acommercial dairy concentrate. The treatments consisted of:

[0088] Treatment 1—Control diet (TMR).

[0089] Treatment 2—TMR containing 0.2% (w/w) Tween 80, and

[0090] Treatment 3—TMR containing 0.3% (w/w) Tween 80.

[0091] The experiment lasted 12 weeks. All cows were fed the controldiet during the first week. This period served as a pretrial week. Cowsin each treatment group were then fed their experimental diets for tenweeks. Milk production and feed intake were, monitored from the firstweek (pretrial) until the 12th week (one week after the experimentaldiets were withdrawn).

[0092] Ambient temperatures exceeded 40° C. during weeks 10 and 11 ofthe experiment resulting in considerable heat stress in the cows. Milkproduction and feed intake data are discussed in the light of the heatstress.

[0093] Average milk production by all animals in each treatment group isdepicted in FIG. 5. Prior to the incidence of heat stress, cows on thetreatment containing 0.2% Tween 80 produced about 1.1 kg/day (3%) moremilk than cows on the control diet. During the first week of the heatstress (week 10), milk production by cows on the control diet fell by anaverage of 13.6%, while that of cows on the Tween 80 treatments fell byabout 11%. The drop in milk production increased to 31.9% in cows on thecontrol diet during week 11, compared to 23.6% in cows that received0.2% Tween 80, and 22% in cows that received 0.3% Tween 80.

[0094] Milk production of first calf heifers in each treatment groupthat had calved at least 21 days prior to the start of the trial revealthat on average, animals on the dietary treatment containing 0.2% Tween80 produced 2 kg/day more milk than animals on the control diet (FIG.6). This number increased to more than 3.6 kg/day during the second weekof heat stress, an increase of 13.6%. Mature cows (cows in secondlactation or greater) on the treatment containing 0.2% Tween 80 produced3.54 kg/day more milk on average and on the treatment containing 0.3%Tween 80 produced 3.98 kg/day more milk on average than animals on thecontrol diet (FIG. 7).

[0095] Dry matter intake by cows on the control diet also fell by 17.4and 30.3% during the first and second week of heat stress. Therespective depressions in dry matter intake were 3.4 and 14.4% in cowson 0.2% Tween 80, and 6.0 and 12.6% in cows on 0.3% Tween 80. Theseresults indicate the ability of Tween 80 to mitigate the effect of heatstress on feed intake and milk production.

[0096] Animals on the control treatment lost about 3.5 kg in weightduring the first 30 d of the experiment and 1 kg during the last 60 d ofthe experiment. Cows on 0.2% Tween 80, however, gained 1 kg during thefirst 30 d and 9 kg during the last 60 days of the experiment. Therespective weight gains in cows on 0.3% Tween 80 were 2.5 and 4.5 kg.This is an indication that the extra milk produced by these animals wasnot derived from mobilization of body reserves.

EXAMPLE 6 Effect of Tween 80 on performance of feedlot cattle

[0097] Three hundred and twenty six Red Angus steers were stratified byweight and divided into eight pens. The pens were then randomly assignedto one of the following four dietary treatments:

[0098] 1) control

[0099] 2) 0.1% (wt/wt) Tween80

[0100]3) 0.2% (wt/wt) glycanase enzyme (enzyme)

[0101] 4) 0.01% Tween 80+0.2% enzyme.

[0102] The enzyme is marketed by GNC Bioferm Inc., Saskatoon, SK. Theproduct contained the following activities (expressed as nmol ofreducing sugars released from 1 mg of product per min: xylanase (336.6),β-glucanase (196.0), carboxymethylcellulase (44.4), and amylase (46.3).The basal diet was a total mixed ration consisting of rolled barley,corn silage and canola meal. Tween 80 was diluted with tap water (1 in5) before it was applied. The total amount of feed required each day forthe animals on each treatment was weighed separately in a mixer wagonand the appropriate quantity of Tween 80, enzyme, or their combinationapplied to it and mixed for ten minutes before feeding. An equal volumeof water as applied to the Tween 80 treatment was also applied to thecontrol and enzyme treatment to make the moisture content of the fourexperimental diets equal. The experimental diets were fed for a total of119 days. Individual body weights were taken at the beginning and end ofthe experiment. Group body weights were taken at monthly intervals.

[0103] Overall body weight changes and feed efficiency in animals oneach of the dietary treatments are indicated in Table 3.1 below. At theend of the 119 days, animals that consumed diets containing 0.1% Tween80 had gained approximately 5.8% more weight than animals on the controldiet. Average daily gain in these animals was 6.3% higher than inanimals consuming the control diet. Feed efficiency was also better inanimals on the 0.1% Tween 80 treatment. TABLE 6 Average daily gain andfeed efficiency in steers fed Tween 80 and enzyme for 119 days¹ InitialBody Total Average Feed Weight Weight Daily Gain Efficiency Treatment²(kg) Gain (kg) (kg/d) (Gain/Feed) Control 422.98 207.03b 1.59b 0.1600.1% Tween 80 427.97 219.03a 1.69a 0.166 0.2% Enzyme 430.24 211.66b1.63ab 0.163 0.01% Tween 80 + 425.47 207.53b 1.60b 0.160 0.2% Enzyme

[0104] 1 Means in the same column with different superscripts differ(P<0.05)

[0105] 2 Concentrations are on dry matter basis.

REFERENCES

[0106] 1. Beauchemin, K. A. et al., “Fibrolytic enzymes increase fiberdigestibility and growth rate of steers fed dry forages,” Can. J. Anim.Sci. 75:641-644 (1995).

[0107] 2. Castanon et al., “Effects of the surfactant Tween 80 onenzymatic hydrolysis of newspaper,” Biotechnol. & Bioeng. 23:1365(1981).

[0108] 3. Cheng et al., “Microbial ecology and physiology of feeddegradation within the rumen,” in Physiological aspects of digestion andmetabolism in ruminants: Proceedings of the seventh internationalsymposium on ruminant physiology, Tsuda, Ed., 1991.

[0109] 4. Feng, P. et al., “Effect of enzyme additives on in situ and invitro degradation of mature cool-season grass forage,” J. Anim. Sci. 70(Suppl. 1): 309 (1992).

[0110] 5. Forsberg, “Some Effects of Arsenic on the Rumen Microflora; AnIn Vitro Study,” Can. J. Microbiol. 24:36 (1978).

[0111] 6. Goering et al., “Forage fiber analysis,” Agric. Handbook No.379, p. 12 (1970).

[0112] 7. Griffin et al., “Surface Active Agents,” in Handbook of FoodAdditives. 2^(nd) Ed., T. E. Furia, Ed., CRC Press, New York, N.Y., p397 et seq. (1972).

[0113] 8. Jouany, J. P, “Methods of manipulating the microbialmetabolism in the rumen,” Ann. Zootech. 43:49-62 (1994).

[0114] 9. Mahadevan et al., “Preparation of protease from mixed rumenmicroorganisms and its use for the in vitro determination of trueprotein in feedstuffs,” Can. J. Anim. Sci 67:55 (1987).

[0115] 10. Parkinson and Allen, “A wet oxidation procedure suitable forthe determination of nitrogen and mineral nutrients in biologicalmaterials,” Comm. Soil Sci. Plant Anal. 6:1 (1975).

[0116] 11. Perry, T. W. et al., “Effects of supplemental enzymes onnitrogen balance, digestibility of energy and nutrients and on growthand feed efficiency of cattle,” J. Anim. Sci. 25:760-764 (1966).

[0117] 12. Rosen, “A Modified Colorimetric Analysis For Amino Acids,”Arch. Biochim. Biophys. 67:10 (1957).

[0118] 13. Sasago et al., “Determination of sulfhydryl and disulfidegroups in milk by p-chloromercuribenzoate-diathizone method,” J. DairySci. 46:1348-1351 (1963).

[0119] 14. Smith et al., “Measurement of protein using bicinchonicacid,” Anal. Biochem. 150:76 (1984).

[0120] 15. Svozil, B. et al., “Application of a cellulolytic preparationin nutrition of lambs,” Sbor. Ved. Praci. VUVZ Prhrelice 22:69-78(1989).

[0121] 16. Uden et al., “Investigation of chromium, cerium and cobalt asmarkers in digesta: Rate of Passage studies,” J. Sci. of Food andAgriculture 31:625-632 (1980).

[0122] 17. Van Nevel, C. J. et al., “Manipulation of rumenfermentation,” In: The Rumen Microbial Ecosystem. Ed. P. N. Hobson.Elsevier Applied Science London. Pp. 387 (1988).

[0123] While the preferred embodiment of the invention has beenillustrated and described, it will be appreciated that various changescan be made therein without departing from the spirit and scope of theinvention.

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. A method of enhancingfeed utilization efficiency in a ruminant animal, comprising adding tothe feed a sufficient amount of a nonionic surfactant to enhance theutilization of the feed by the animal.
 2. A method of claim 1 whereinthe nonionic surfactant is selected from the group consisting ofpolyoxyethylenesorbitan monooleate, polyoxyethylenesorbitan trioleate,polyoxyethylenesorbitan monostearate, alkyltrimethylammonium bromides,dodecyltrimethylammonium bromide, hexadecyltrimethylammonium bromide,mixed alkyltrimethylammonium bromide, tetradecyltrimethylammoniumbromide, benzalkonium chloride, benzethonium chloride,benzyldimethyldodecylammonium bromide, benzyldimethylhexadecylammoniumbromide, benzyltrimethylammonium chloride, benzyltrimethylammoniummethoxide, cetylpyridinium bromide, cetylpyridinium chloride,cetyltributylphosphonium bromide, cetyltrimethylammonium bromide,decamethonium bromide, dimethyldioctadecylammonium bromide,methylbenzethonium chloride, methyl mixed trialkyl ammonium chloride,methyltrioctylammonium chloride,n,n′,mb′-polyethylene(10)-n-tallow-1,3-diamino-propane and 4-picolinedodecyl sulfate.
 3. A method of claim 2 wherein the nonionic surfactantis selected from the group consisting of polyoxyethylenesorbitanmonooleate and polyoxyethylenesorbitan trioleate.
 4. A method of claim 2wherein the nonionic surfactant comprises from about 0.01 to 1% (w/w) ofthe dry weight of the feed.
 5. A method of claim 4 wherein the nonionicsurfactant comprises from about 0.01 to 0.3% (w/w) of the dry weight ofthe feed.
 6. A method of claim 1 wherein the nonionic surfactant isadded to the feed by spraying a dilute liquid solution comprising thesurfactant onto the feed.
 7. A method of claim 1 wherein the nonionicsurfactant is added to the feed by coating the nonionic surfactant ontoa solid particulate carrier and then adding the carrier to the feed. 8.A method of claim 7 wherein the solid particulate carrier is selectedfrom the group consisting of celite, diatomaceous earth and silica
 9. Amethod of claim 1 which further comprises adding a digestion enhancingenzyme to the feed.
 10. A method of claim 1 which further comprisesadding a lactic acid bacteria inoculum to the feed.
 11. A method ofenhancing weight gain in a ruminant animal, comprising adding to thefeed of the animal a sufficient amount of a nonionic surfactant toenhance weight gain by the animal.
 12. A method of claim 11 wherein thenonionic surfactant is selected from the group consisting ofpolyoxyethylenesorbitan monooleate, polyoxyethylenesorbitan trioleate,polyoxyethylenesorbitan monostearate, alkyltrimethylammonium bromides,dodecyltrimethylammonium bromide, hexadecyltrimethylammonium bromide,mixed alkyltrimethylammonium bromide, tetradecyltrimethylammoniumbromide, benzalkonium chloride, benzethonium chloride,benzyldimethyldodecylammonium bromide, benzyldimethylhexadecylammoniumbromide, benzyltrimethylammonium chloride, benzyltrimethylammoniummethoxide, cetylpyridinium bromide, cetylpyridinium chloride,cetyltributylphosphonium bromide, cetyltrimethylammonium bromide,decamethonium bromide, dimethyldioctadecylammonium bromide,methylbenzethonium chloride, methyl mixed trialkyl ammonium chloride,methyltrioctylammonium chloride,n,n′,mb′-polyethylene(10)-n-tallow-1,3-diamino-propane and 4-picolinedodecyl sulfate.
 13. A method of claim 12 wherein the nonionicsurfactant is selected from the group consisting ofpolyoxyethylenesorbitan monooleate and polyoxyethylenesorbitantrioleate.
 14. A method of claim 12 wherein the nonionic surfactantcomprises from about 0.01 to 1% (w/w) of the dry weight of the feed. 15.A method of claim 14 wherein the nonionic surfactant comprises fromabout 0.01 to 0.3% (w/w) of the dry weight of the feed.
 16. A method ofclaim 11 wherein the nonionic surfactant is added to the feed byspraying a dilute liquid solution comprising the surfactant onto thefeed.
 17. A method of claim 11 wherein the nonionic surfactant is addedto the feed by coating the nonionic surfactant onto a solid particulatecarrier and then adding the carrier to the feed.
 18. A method of claim17 wherein the solid particulate carrier is selected from the groupconsisting of celite, diatomaceous earth and silica
 19. A method ofclaim 11 which further comprises adding a digestion enhancing enzyme tothe feed.
 20. A method of claim 11 which further comprises adding alactic acid bacteria inoculum to the feed.
 21. A method of enhancingmilk production by a ruminant animal, comprising adding to the feed ofthe animal a sufficient amount of a nonionic surfactant to enhance milkproduction by the animal.
 22. A method of claim 21 wherein the nonionicsurfactant is selected from the group consisting ofpolyoxyethylenesorbitan monooleate, polyoxyethylenesorbitan trioleate,polyoxyethylenesorbitan monostearate, alkyltrimethylammonium bromides,dodecyltrimethylammonium bromide, hexadecyltrimethylammonium bromide,mixed alkyltrimethylammonium bromide, tetradecyltrimethylammoniumbromide, benzalkonium chloride, benzethonium chloride,benzyldimethyldodecylammonium bromide, benzyldimethylhexadecylammoniumbromide, benzyltrimethylammonium chloride, benzyltrimethylammoniummethoxide, cetylpyridinium bromide, cetylpyridinium chloride,cetyltributylphosphonium bromide, cetyltrimethylammonium bromide,decamethonium bromide, dimethyldioctadecylammonium bromide,methylbenzethonium chloride, methyl mixed trialkyl ammonium chloride,methyltrioctylammonium chloride,n,n′,mb′-polyethylene(10)-n-tallow-1,3-diamino-propane and 4-picolinedodecyl sulfate.
 23. A method of claim 22 wherein the nonionicsurfactant is selected from the group consisting ofpolyoxyethylenesorbitan monooleate and polyoxyethylenesorbitantrioleate.
 24. A method of claim 22 wherein the nonionic surfactantcomprises from about 0.01 to 1% (w/w) of the dry weight of the feed. 25.A method of claim 24 wherein the nonionic surfactant comprises fromabout 0.01 to 0.3% (w/w) of the dry weight of the feed.
 26. A method ofclaim 21 wherein the nonionic surfactant is added to the feed byspraying a dilute liquid solution comprising the surfactant onto thefeed.
 27. A method of claim 21 wherein the nonionic surfactant is addedto the feed by coating the nonionic surfactant onto a solid particulatecarrier and then adding the carrier to the feed.
 28. A method of claim27 wherein the solid particulate carrier is selected from the groupconsisting of celite, diatomaceous earth and silica
 29. A method ofclaim 21 which further comprises adding a digestion enhancing enzyme tothe feed.
 30. A method of claim 21 which further comprises adding alactic acid bacteria inoculum to the feed.
 31. A feedstuff compositionfor ruminant animals comprising a feedstuff and a sufficient amount of anonionic surfactant to enhance the utilization of the feedstuff by theanimal.
 32. A composition of claim 31 wherein the nonionic surfactant isselected from the group consisting of polyoxyethylenesorbitanmonooleate, polyoxyethylenesorbitan trioleate, polyoxyethylenesorbitanmonostearate, alkyltrimethylammonium bromides, dodecyltrimethylammoniumbromide, hexadecyltrimethylammonium bromide, mixedalkyltrimethylammonium bromide, tetradecyltrimethylammonium bromide,benzalkonium chloride, benzethonium chloride,benzyldimethyldodecylammonium bromide, benzyldimethylhexadecylammoniumbromide, benzyltrimethylammonium chloride, benzyltrimethylammoniummethoxide, cetylpyridinium bromide, cetylpyridinium chloride,cetyltributylphosphonium bromide, cetyltrimethylammonium bromide,decamethonium bromide, dimethyldioctadecylammonium bromide,methylbenzethonium chloride, methyl mixed trialkyl ammonium chloride,methyltrioctylammonium chloride,n,n′,mb′-polyethylene(10)-n-tallow-1,3-diamino-propane and 4-picolinedodecyl sulfate.
 33. A composition of claim 32 wherein the nonionicsurfactant is selected from the group consisting ofpolyoxyethylenesorbitan monooleate and polyoxyethylenesorbitantrioleate.
 34. A composition of claim 32 wherein the nonionic surfactantcomprises from about 0.01 to 1% (w/w) of the dry weight of thefeedstuff.
 35. A composition of claim 34 wherein the nonionic surfactantcomprises from about 0.01 to 0.3% (w/w) of the dry weight of thefeedstuff.
 36. A composition of claim 31 wherein the nonionic surfactantis added to the feedstuff by spraying a dilute liquid solutioncomprising the surfactant onto the feed.
 37. A composition of claim 31wherein the nonionic surfactant is added to the feedstuff by coating thenonionic surfactant onto a solid particulate carrier and then adding thecarrier to the feedstuff.
 38. A composition of claim 37 wherein thesolid particulate carrier is selected from the group consisting ofcelite, diatomaceous earth and silica
 39. A composition of claim 31which further comprises adding a digestion enhancing enzyme to thefeedstuff.
 40. A composition of claim 31 which further comprises addinga lactic acid bacteria inoculum to the feedstuff.